Microscope, particularly a laser scanning microscope with adaptive optical arrangement

ABSTRACT

A microscope, particularly a laser scanning microscope, with an adaptive optical device in the microscope beam path, comprising two reflective adaptive elements, at least one of which is constructed as an adaptive optical element, both of which are oriented with their reflector surface vertical to the optical axes of the microscope beam path, and a polarizing beam splitter whose splitter layer is located in the vertex of two orthogonal arms of the microscope beam path or two orthogonal portions of a folded microscope beam path, wherein a first adaptive element is associated with one arm and the other adaptive element is associated with the second arm, and a quarter-wave plate is located in each arm between the beam splitter and reflective adaptive element, and a detection device to which the detection light is directed and which is linked to the adaptive elements by evaluating and adjusting devices.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority of German Application No. 102 27 120.8,filed Jun. 15, 2002, the complete disclosure of which is herebyincorporated by reference.

BACKGROUND OF THE INVENTION

a) Field of the Invention

The invention is directed to a microscope, particularly a laser scanningmicroscope, with an adaptive optical device for variable adaptation ofthe illumination beam path and/or detection beam path to thecharacteristics of the optical component groups and/or to thecharacteristics of the sample.

b) Description of the Related Art

The use of adaptive optics in microscopes is already known, per se, inthe prior art and is described, for example, in PCT/WO99/06656. However,the use of the adaptive optics in the illumination beam path and/ordetection beam path as is disclosed therein requires the use of at leastone beam splitter which must be incorporated in the design resulting inoptical losses.

Coupling adaptive elements into the beam path without losses by means ofmirrors as is shown in principle in the accompanying drawing labeled“prior art” is also known, but leads to additional substantialaberrations due to the oblique incidence of the beam path on thereflector faces.

Further, the prior art requires interaction between the adaptive opticalelements and a wavefront sensor, so that further losses occur ingeneral.

Formerly conventional sensors such as Shack-Hartmann sensors orinterferometers are used as wavefront sensors in this connection as isindicated, for example, in PCT/GB99/03194. However, sensors of this kindare not well suited to three-dimensional scanning of a sample as isintended with a confocal laser scanning microscope. This is due to thefact that, in contrast to confocal detection, a relatively large amountof unfocused light strikes the sensor.

OBJECT AND SUMMARY OF THE INVENTION

Proceeding from this prior art, the invention has the primary object offurther developing a microscope of the type mentioned in the beginningin such a way that a loss-free and aberration-free coupling of theadaptive optics into the illumination beam path and/or detection beampath is ensured. Another object of the invention is to realize asuitable regulation of the adaptive optics without the use of specialwavefront sensors.

According to the invention, the adaptive optical device comprises

-   -   two reflective adaptive elements, both of which are oriented        with their reflector surface vertical to the optical axis of the        microscope beam path, and a polarizing beam splitter whose        splitter layer is located in the vertex of two orthogonal        branches or arms of the microscope beam path, wherein a first        adaptive element is associated with one arm and the other        adaptive element is associated with the second arm, and a        quarter-wave plate is located in each of the arms between the        beam splitter and adaptive element, and    -   a detection device to which the detection light is directed and        which is linked to the adaptive element by evaluating and        adjusting devices.    -   The detection device which exists in the laser scanning        microscope anyway should preferably be used as detection device.

Depending on the evaluation and processing of the detector signals, itis possible with this arrangement to achieve fast depth scanning in thethree-dimensional scanning of a sample without a displacing movement ofthe microscope objective or the sample, to compensate for aberrationscaused by the sample and also to correct aberrations caused byincorrectly used or incompletely corrected optical components,particularly the microscope objective.

In a construction of the invention, a polarizing beam splitter is usedand a quarter-wave plate is located in each of the arms between the beamsplitter and adaptive element. In this arrangement, the light comingfrom the illumination source is directed in the one arm through thesplitter layer and a quarter-wave plate to the first adaptive elementdepending on its polarization state, is reflected back from thereflector surface of the adaptive element to the splitter layer and ispropagated in the direction of the microscope objective. The light isdeflected in the direction of the other arm with orthogonal polarizationwith respect to the light propagating in the direction of the firstadaptive element, a second adaptive element being located at the end ofthe other arm. The light is reflected back at this element after passingthrough a quarter-wave plate to the splitter layer and passes throughthe latter to the microscope objective.

The detection light radiated from the sample and coming through themicroscope objective passes through one arm or the other arm dependingon its polarization state, is reflected back to the splitter layer bythe corresponding reflective adaptive element and propagates toward thedetection device.

The detection device has a second polarizing beam splitter and twooptoelectronic converters. Depending on its polarization state, thedetection light passes through the splitter layer of this beam splitterto the one optoelectronic converter or is deflected by the splitterlayer to the other optoelectronic converter. The signal from theconverters can be uniquely correlated to the light traveling on thedetection side via a determined arm of the beam splitter arrangementwith the adaptive elements.

The essential advantage of this arrangement consists in that it makes itpossible to couple the adaptive optics into the illumination beam pathand/or detection beam path of the microscope without losses oraberrations. Further, there are different advantageous modes ofregulation of the adaptive optics as will be explained more fully in thefollowing. The arrangement according to the invention can be used in apoint-scanning laser scanning microscope or in a line-scanning laserscanning microscope.

Pinhole optics for focusing the detection light on a pinhole and opticsfor collimating the detection light are provided between the beamsplitter associated with the adaptive elements and the detection device,corresponding to the construction of a confocal laser scanningmicroscope.

The object of the invention is also met by an adaptive optical devicewhich comprises the following:

-   -   an adaptive reflective element and a nonadaptive reflective        element, both of which are oriented with their reflector surface        vertical to the optical axis of the microscope beam path, and a        polarizing beam splitter whose splitter layer is located in the        vertex of two orthogonal arms of the microscope beam path,        wherein the adaptive element is associated with one arm and the        nonadaptive element is associated with the second arm, and a        quarter-wave plate is located in each arm between the beam        splitter and the respective reflective element, and    -   a detection device to which the detection light is directed and        which is linked to the adaptive element via an evaluating and        adjusting device.

The existing detection device of the laser scanning microscope shouldpreferably be used as detection device.

An arrangement can be provided in which the light coming from theillumination source is directed in one arm through the splitter layerand a quarter-wave plate to the adaptive element depending on itspolarization state, is reflected back to the splitter layer by thereflector surface of the adaptive element and is propagated in thedirection of the microscope objective.

The light with orthogonal polarization with respect to the lightpropagating in the direction of the first adaptive element is guided inthe direction of the other arm, a nonadaptive element (a mirror) beinglocated at the end of the other arm. At this mirror, the light isreflected back to the splitter layer after passing through aquarter-wave plate and passes through the splitter layer to themicroscope objective.

In this construction, it is possible to do without the adaptive elementon the excitation side by suitable polarization of the light and toobtain uncorrected imaging in combination with the detection of thesignal from the nonadaptive element or to switch quickly betweencorrected and uncorrected imaging by rotating the polarization.

In both of the constructions mentioned above, the adaptive device can beprovided in the excitation beam path and detection beam path as isdescribed in the first arrangement or only in the excitation beam path.The latter is particularly suitable for use in connection withmultiphoton excitation, wherein the adaptive optical device is placed onthe excitation side prior to the separation of the illumination beampath and detection beam path.

The adaptive elements are preferably positioned in a pupil plane of themicroscope beam path.

The pupil plane is advantageously identical to the reflecting surfacesof the scanning device and the scanning optics, the microscope objectiveand a tube lens are positioned relative to one another in such a waythat a diffraction-limited spot is generated in the sample, which spotis guided over the sample in lateral direction due to the scanningmovement. As has already been stated, point scanning or line scanningcan be carried out.

For example, mirrors with mirror surfaces that are adjustable insegments, diaphragm mirrors or reflective spatial light modulators canbe used as adaptive reflective elements.

Another preferred construction of the arrangement according to theinvention consists in that zoom optics are associated with the adaptiveelements and serve to adapt the aperture of the respective adaptiveelement to the aperture of the microscope objective.

Another alternative constructional variant comprises:

-   -   an adaptive concave mirror which is arranged in a pupil plane of        the illumination beam path, an optical beam splitter being        associated with the adaptive concave mirror in an intermediate        image plane and having a transmittive area and a reflective area        in a splitter surface, wherein the illumination light is        directed initially to the splitter surface and from the        reflective area of the latter to the concave mirror, is focused        by the concave mirror onto the transmittive area and passes        through the latter to the sample and/or    -   an adaptive concave mirror which is arranged in a pupil plane of        the detection beam path, an optical beam splitter being        associated with the adaptive concave mirror in an intermediate        image plane and having a transmittive area and a reflective area        in a splitter surface, wherein the detection light is directed        initially to the splitter surface and from its reflective area        to the concave mirror, is focused by the concave mirror onto the        transmittive area and passes through the latter to a detection        device, and    -   an evaluating device which is connected on the input side to the        detection device of the microscope and on the output side to        adjusting devices for varying the shape of one or both concave        mirrors and/or for changing the distance between the respective        concave mirror and the associated beam splitter.

With this arrangement, the adaptive concave mirror in the illuminationbeam path can again be used for fast focusing or for correcting imageerrors which are caused by the optical component groups or by the sampleto be examined. The transmittive area of the splitter surface does notact as a confocal diaphragm or spatial filter, and it has a diameter atleast greater than 5 Airys.

The detection light coming from the sample is branched off from theillumination beam path by a beam splitter which is provided for thispurpose and which is preferably positioned between collimating opticsfor the illumination light and the scanning device.

When a point-scanning device is provided for scanning the sample, thetransmittive area of the splitter surface is formed as a circular orelliptical opening. The elliptically shaped opening is advantageous inthat there is a circular projection in the direction of the concavemirror when the splitter surface is inclined by 45° relative to theincident radiation.

In case of a line-scanning device for scanning the sample, thetransmittive area of the splitter surface is formed as a slit-shapedopening.

Further, it can be advantageous when the illumination light and/or thedetection light which pass(es) through the transmittive area of theassociated splitter surface are/is followed by a detector on which thebeam component falling through impinges and which serves for evaluatingthe beam intensity of this light.

All of the adaptive concave mirrors provided in the arrangementaccording to the invention can also be coupled with a device fordisplacing them in the direction of the optical axis, wherein thisdisplacement device likewise communicates with the evaluating andadjusting device, and wherein the displacement of the adaptive concavemirror and the adjustment of its focal length influence the focusing inthat the geometry of the mirror surface is changed by correspondingcontrol. Among other things, the aperture of the concave mirror isadapted to the pupils of the used microscope objectives in this way.

Apart from the almost loss-free and aberration-free coupling of theadaptive element into the microscope beam path, the particular advantageof the latter design consists in the ideal chromatic correction bydispensing with refractive elements.

The reflective adaptive elements mentioned above can be used as adaptiveconcave mirrors.

Further, refractive elements can also be associated with the adaptiveconcave mirrors for reducing their focal lengths.

In addition, a device can be provided which makes it possible to swivela wavelength filter into the detection beam path in order to suppressthe illumination light, particularly with fluorescence detection.

Further, relay optics can also be arranged in the beam path and can beused to generate a pupil at the location of an adaptive concave mirror.

The invention will be explained more fully in the following withreference to embodiment examples.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. A shows the state of the art in schematic form;

FIG. 1 shows the beam path of a confocal laser scanning microscope withan adaptive optical device, according to the invention, comprising twoadaptive mirrors;

FIG. 2 shows a constructional variant of the beam path of a confocallaser scanning microscope with an adaptive optical device, according tothe invention, comprising an adaptive mirror and a nonadaptive mirror;

FIGS. 3 a and 3 b show a schematic view of another embodiment example ofthe arrangement, according to the invention, with a beam splitter whichhas a reflective area and a transmittive area;

FIG. 4 shows measurement values obtained by iteration during the imagingof a biological sample with the arrangement according to the invention;and

FIG. 5 shows an example of possibilities for correction during theimaging of a biological sample using the arrangement according to theinvention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In FIG. 1 the arrangement according to the invention is illustrated withreference to a beam path of a confocal laser scanning microscope inwhich the illumination light 2 proceeding from an illumination source 1is directed to a sample 3. In so doing, a diffraction-limited spot isgenerated by scanning optics 4, a tube lens 5 and the microscopeobjective 6, this spot being moved over the sample 3 in lateraldirection by means of a scanning device 7 for obtaining images.

The light emitted by the sample 3 passes through the microscopeobjective 6, tube lens 5 and scanning optics 4 in the opposite directionand is directed to a detection device 10 via a beam splitter 8 whichserves to divide the detection beam path 9 and the illumination light 2.Pinhole optics 11, a pinhole 12 and collimating optics 13 are arrangedfollowing the beam splitter 8 in the detection beam path 9.

Further, a wavelength filter 14 which prevents illumination light 2 fromreaching the detection device 10 particularly in fluorescence detectionis provided.

According to the invention, there is a first adaptive mirror 15 whosereflector surface is oriented vertical to the optical axis of an arm 16of the microscope beam path. Further, another adaptive mirror 17 isoriented with its reflector surface vertical to the optical axis of anarm 18 of the microscope beam path.

A polarizing beam splitter 19 whose splitter layer is inclined by 45°relative to the beam direction in both arms 16, 18 is arranged in thevertex of the angle enclosing the two arms 16 and 18.

Further, a quarter-wave plate 20 and zoom optics 21 are provided betweenthe beam splitter 19 and the adaptive mirror 15, and a quarter-waveplate 22 and zoom optics 23 are provided between the beam splitter 19and the adaptive mirror 17.

Finally, a lens 24 whose purpose is to image the planes of the adaptivemirrors 15 and 17 in a pupil plane 25 of the microscope arrangement isprovided. However, this function can also be taken over simultaneouslyby the zoom optics 21 or 23 when suitably designed, in which case thelens 24 is dispensed with.

During the scanning of the sample 3, after the illumination light 2 isdeflected in beam splitter 8, depending on its polarization state, itpasses through the splitter layer of beam splitter 19 to the adaptivemirror 15 or, deflected by the splitter film, to the adaptive mirror 17or also to both adaptive mirrors 15, 17.

The light is reflected back again to the beam splitter 19 by theadaptive mirror 15 or 17 on which it impinges. The illumination lightwhich travels back to the beam splitter 19 after passing twice throughthe quarter-wave plate 20 or 22 is polarized orthogonal to the incidentlight. Consequently, the illumination light is coupled into themicroscope beam path again by way of the splitter layer of the beamsplitter 19 and is then directed to the sample 3.

This in-coupling is carried out without losses due to the adaptivedevice according to the invention which comprises the adaptive mirrors15, 17, the polarizing beam splitter 19 and the quarter-wave plate 20,22.

The light emitted by the sample 3 traverses the light path in thereverse sequence via the beam splitter 19 and adaptive mirrors 15, 17.Then, depending on its polarization state, the detection light is eitherdeflected by the splitter layer of the beam splitter 19 to the adaptivemirror 15 or travels through the splitter layer of the beam splitter 19to the adaptive mirror 17 or to both adaptive mirrors 15, 17. Thedetection light is also thrown back again by the adaptive mirrors 15, 17and is polarized orthogonal to the arriving detection light afterpassing twice through the quarter-wave plates 20 and 22. This means thatthe light coming to the adaptive mirror 15 and reaching the detectiondevice 10 is polarized orthogonal to the light coming from adaptivemirror 17.

On this basis, a polarizing beam splitter 26 and two optoelectronicconverters 27.1, 27.2 are provided in the detection device 10.

Accordingly, it is possible to detect the detection light coming fromadaptive mirror 15 by means of the optoelectronic converter 27.1 and todetect the detection light coming from adaptive mirror 17 by means ofoptoelectronic converter 27.2; that is, signals which are separatelyassociated with the adaptive mirrors 15 and 17, respectively, on thedetection side are applied to the outputs of the optoelectronicconverter 27.1, 27.2.

According to the invention, it is further provided that the outputsignal of the optoelectronic converter 27.1 communicates with theadaptive mirror 15 via an evaluating and adjusting device, and theoutput of the optoelectronic converter 27.2 likewise communicates withthe adaptive mirror 17 by means of an evaluating and adjusting device.

Further, the zoom devices 21 and 23 are also coupled with the respectiveassociated evaluating and adjusting device.

With the arrangement according to the invention, it is possible to usethe adaptive optics for fast focusing or for correcting image errorscaused by the optical component groups or by the sample to be examinedwith high efficiency and with relatively small expenditure on apparatus.The corresponding methods are described further below.

In the following, another embodiment example will be described withreference to FIG. 2. The illumination light 29 proceeding from anillumination source 28 is again directed by means of scanning optics 30,a tube lens 31 and the microscope objective 32 to a sample 33. Adiffraction-limited spot is guided over the sample 33 in lateraldirection by means of a scanning device 34 and image information of thesample 33 is obtained in this way.

The light radiated by the sample 33 traverses the microscope objective32 and the tube lens 31 in the reverse direction, is split from theillumination beam path by means of a beam splitter 35 and is thendirected to a detector 38 by pinhole optics 37 as a separate detectionbeam path 36.

According to the invention, an adaptive mirror 39 and a conventional,nonadaptive mirror 40 are provided in the beam path of the illuminationlight 29 between the illumination source 28 and the scanning device 34and are oriented with their reflector surfaces vertical to two arms 41,42 of the illumination beam path. The two arms 41, 42 enclose a rightangle in whose vertex the splitter surface of a polarizing beam splitter43 is located. The splitter surface is inclined by 45° to the beamdirection in the two arms 41, 42.

Further, a quarter-wave plate 44 is arranged in the beam path betweenthe beam splitter 4 and the mirror 40, and a quarter-wave plate 45 andzoom optics 46 are arranged in the beam path between the adaptive mirror39 and the beam splitter 43.

When this microscope arrangement is operated, the illumination light 29passes through the splitter surface of the beam splitter 43 via thequarter-wave plate 45 and the zoom optics 46 to the adaptive mirror 39depending on its polarization state in a manner analogous to thepreceding embodiment example, and is reflected back by the adaptivemirror 39 over the same path to the splitter surface of the beamsplitter 43. A deflection takes place from the splitter surface towardthe sample 33, since the illumination light 29 is now polarizedorthogonal to the incident illumination light 29 after passing twicethrough the quarter-wave plate 45.

When the polarization diverges, the illumination light 29 is initiallydeflected by the splitter layer of the beam splitter 43 toward themirror 40 and is reflected back from the latter to the splitter layer,passes through the quarter-wave plate 44 twice and, after passingthrough the quarter-wave plate 44 twice, has a polarization directionorthogonal to the incident illumination light 29, as a result of whichthe illumination light now passes through the splitter layer to thesample 33.

The light radiated from the sample 33 reaches the detector 38 along thepath described above, the detector 38 communicates with the adaptivemirror 39 and/or the zoom device 46 via an evaluating and adjustingdevice, not shown. The procedure when this arrangement is used for imagegeneration will be explained more exactly below.

The actuating signals which are generated in the evaluating device forthe zoom optics 46 can serve to adapt the beam expansion to themicroscope objective in an optimal manner.

FIG. 3 a shows a schematic view of another embodiment example of thearrangement according to the invention. In this case, the illuminationlight 49 proceeding from an illumination source 48 strikes the splittersurface 50 of an optical beam splitter.

It can be seen in FIG. 3 b that the splitter surface 50 has atransmittive area 51 formed as a circular opening which is enclosed by areflective area 52. Alternatively, the transmittive area 51 can also beformed as an elliptical opening. As is further shown in FIG. 3 a, thesplitter surface 50 is inclined by 45° relative to the incidentillumination light 49. In this way, the beam component 49.1 of theillumination light 49 striking the reflective area 52 is deflected to anadaptive concave mirror 53 which is located in a pupil plane of themicroscope arrangement.

The adaptive concave mirror 53 focuses the beam component 49.1 in itselfback to the transmittive area 51 located in an intermediate image planeof the arrangement and accordingly acts as a confocal diaphragm in theillumination beam path.

In this connection, it is possible to influence or adjust the opticalresolution of the microscope arrangement through the use of splittersurfaces with transmittive areas of different diameter.

When the transmittive area 51 has an elliptical shape, this shape takeson an apparently circular shape because of the 45-degree inclination ofthe splitter surface 50 for the radiation coming from the concave mirror53.

The beam component 49.1 focused on the transmittive area 51 passesthrough the transmittive area 51 and is subsequently collimated througha lens 54. A diffraction-limited spot serving for recording images ofthe sample 58 is generated in the sample 58 by means of scanning optics55, a tube lens 56 and the microscope objective 57 and is moved for thispurpose in lateral direction over the sample 58 by means of a scanningdevice 59.

The detection light proceeding from the sample 58 and carrying along theimage information passes on its return path through the microscopeobjective 57, tube lens 56, scanning optics 55 and scanning device 59 toa dichroic beam splitter 60 which separates the detection beam path 61from the illumination beam path and directs it to the splitter surface62 of another beam splitter.

Splitter surface 62 is constructed identical to splitter surface 50 withrespect to geometry and orientation to the detector beam path 61.

Since, consequently, the splitter surface 62 likewise has a reflectivearea 52 (see FIG. 3 b), a predominant proportion 61.1 of the detectionlight is directed to an adaptive concave mirror 63. The concave mirror63 focuses the beam portion 61.1 in itself back to the transmittive area51 and through the latter, whereupon the detection light strikes adetector 64.

The intensity of the detection light is measured by means of thedetector 64 and the measured value is conveyed to an evaluating device,not shown in the drawing, which communicates with adjusting devices forchanging the geometry of the mirror surfaces of the adaptive concavemirrors 53 and 63.

The reflective wavefront in one or both concave mirrors 53, 63 isinfluenced in such a way when the mirror geometry is adjusted that anoptimal correction of aberrations is achieved and a determineddefocusing is adjusted.

In another construction of this embodiment example, another detector 65,for example, a monitor diode, can be arranged following the splittersurface 50 in the direction of the illumination light 49 and can be usedto monitor the average output of the illumination light.

The quantity of the coupled-out light is given by the function:

$T = {\frac{A_{HT}}{A_{pupil}} = \frac{r_{HT}^{2}}{r_{pupil}^{2}}}$where T is the transmission, A_(HT) is the surface of the transmittivearea, A_(pupil) is the effective pupil cross section, r_(HT) is theradius of the transmittive area and r_(pupil) is the radius of thepupil, where advantageously T˜1%. The radius of the reflective area isadvantageously about 5 mm, the radius of the transmittive area (viewedin the direction of projection or in the direction of radiation) is lessthan 0.25 mm.

A wavelength filter 66 which can be swiveled into the detection beampath 61 can be provided optionally so that the illumination light doesnot reach the detector 64, particularly in fluorescence detection.

Further, it has proven advantageous to arrange relay optics 67 in thedetection beam path 61, which relay optics 67 are used for generating apupil at the location of the adaptive concave mirror 63. They can beconstructed at the same time as zoom optics and enable a variable beamexpansion. The splitter surface 62 is located in an intermediate imageplane of the microscope arrangement and acts as a confocal diaphragm inthe detection beam path 61.

When the diameter of the beam impinging on the splitter surface 62 orthe diameter of the transmittive area 51 of the splitter surface 62changes or splitter surfaces with transmittive areas of differentdiameter are exchanged for one another, the optical resolution of themicroscope arrangement can be influenced or adjusted in this way.

The portion of illumination light or detection light impinging on thetransmittive area 51 is lost at the splitter surfaces 50 and 62 when notin focus. However, the ratio of the surface of the transmittive area 51to the surface of the reflective area 52 is:

$R = {\frac{A_{pupil} - A_{HT}}{A_{pupil}} = \frac{r_{pupil}^{2} - r_{HT}^{2}}{r_{pupil}^{2}}}$where A_(pupil) is the effective pupil cross section, A_(HT) is thesurface of the transmittive area 51, r_(pupil) is the radius of thepupil, r_(HT) is the radius of the transmittive area 51. The radius forthe reflective area 52 for a microscope arrangement is typically about 5mm, while the radius of the transmittive area 51 is less than 0.25 mm.

As a result, the surface ratio and therefore the efficiency of the beamsplitting in the two splitter surfaces 50 and 62 is given by R>99%. Theefficiency achieved in this way is not dependent upon the respectivewavelength of light.

The operation of the arrangement according to the invention will beexplained in the following for different operating modes of a confocallaser scanning microscope. Only the embodiment example according to FIG.1 is used for this description. The arrangements according to the restof the embodiment examples can be applied in an analogous sense.

Reflection Microscopy

In this type of operation, the light that is scattered or reflected bythe sample 3 is used for image generation.

In cases where the sample 3 does not have a birefringent action or doesnot have a pronounced reflectivity dependent upon polarization, thefollowing procedure can be taken. The polarization of the illuminationlight is oriented in such a way that light of the same intensity strikesadaptive mirrors 15 and 17. In the detection beam path 9, the lightreflected by the sample strikes the adaptive mirrors 15 and 17 againwhen passing the two arms 16 and 18, wherein the light striking theconverter 27.1 has been reflected by the adaptive mirror 15 on theexcitation side as well as on the detection side, and the light reachingthe converter 27.2 has been reflected by the adaptive mirror 17 on theexcitation side as well as on the detection side.

As a result of aberrations caused by defocusing, by the sample or byoptical elements, a light intensity recorded at the detectors in theconfocal arrangement is less than that recorded with aberration-freeimaging. The reception signal can be evaluated with respect to aspecific point on the sample or as an average value over a givenscanning surface on the sample. When the geometries of the mirrorsurfaces of the adaptive mirrors 15 and 17 are changed and the effectson the reception signal are measured, a mirror geometry with which thereis an optimal reception signal and with which the optical system istherefore corrected with respect to aberrations can be found andadjusted depending on the measurement results.

In a preferred mode of operation, the geometric shape of the mirrorsurface of the adaptive mirrors 15 and 17 is varied using Zernikepolynomials. For this purpose, the development coefficients (z₁, z₂, . .. z_(i) . . . z_(N)) determine the shape of the mirror surface uniquely.In the present arrangement, the gradient of the intensity of thereception signal with respect to a Zernike coefficient∂I/∂z_(i)≈ΔI/Δz_(i) can be obtained by one measurement in that a mirrorshape (z_(i), z₂, . . . z_(i)+Δz_(i) . . . z_(N)) is adjusted in theadaptive mirror 15 and a mirror shape (z₁, z₂, . . . z_(i) . . . z_(N))is adjusted with adaptive mirror 17.

The standardized gradient is given by grad_(i)=(I_(i) ^(Del1)−I_(i)^(Del2))/Δz_(i)*2/(I_(i) ^(Del1)+I_(i) ^(Del2)), where I_(i) ^(Del1) andI_(i) ^(Del2) are the intensity values measured by the optoelectronicconverters 27.1 and 27.2.

This measurement is carried out for all relevant coefficients. The newmirror shape is given by the gradient from (z₁′, z₂′, . . .z_(N)′)=(z₁+(Δz₁)²grad₁, z₂+(Δz₂)²grad₂, . . .z_(N)+(Δz_(N))²grad_(N))), i.e., every new coefficient is given asz_(i)′=z_(i)+(Δz_(i))²grad_(i).

This procedure is repeated until a determined correction criterion ismet. The step size Δz_(i) can be adapted in a suitable manner. Forexample, FIG. 4 shows the above-described iteration process for imagingat a depth of 200 μm inside a biological specimen with a water immersionobjective with NA=1.2 at a wavelength of 488 nm.

The difference in the index of refraction between the sample and thewater immersion layer is 1.38−1.33=0.05, which leads as a condition ofaberrations to a Strehl ratio of 0.06 (measured intensity divided by theintensity without any aberrations). After about sixty iterations of thetype shown above, including the first and second order sphericalaberration, the optimum of the correction is achieved which leads to aStrehl ratio of 0.92.

For more far-reaching corrections, additional Zernike terms must beincluded. In every iteration step, the gradients for the utilizedcoefficients are measured and influence the mirror geometry as wasalready stated.

When the optimization is begun with a previously estimated correction,there is a considerable acceleration of the iteration process. Theoptimum of the correction can also be achieved in less than 0.3 s at aworking frequency of the mirror adjustment of 0.5 kHz with about sixtyiterations.

Of course, the details of the iteration process can be modified in asimple manner. The description given above is only one embodimentvariant of such a method. In principle, optimization methods ofdifferent types can be used.

In cases where the sample acts in a birefringent manner or itsreflectivity depends extensively on the polarization, it is necessary touse identical settings for the adaptive mirrors 15 and 17. When thereception values of the converters 27.1 and 27.2 are added, thepolarization-dependent effect of the sample is eliminated. Twomeasurements (with mirror position as described (z₁, z₂, . . . z_(i) . .. z_(N)) and (z₁, z₂, . . . z_(i)+Δz_(i)/4 . . . z_(N))) are nowrequired for every coefficient in order to obtain a local gradient. Atthe same time, the difference of the reception signals can be used forpolarization-sensitive measurements.

A special form of phase contrast is Differential Interference Contrast(DIC). This contrast method can be implemented in a simple manner withthe arrangement according to the invention. In this case, theillumination light is divided between the adaptive mirrors 15 and 17with equal intensity. The two adaptive mirrors are used for tilting thebeam of one polarization relative to the beam of the other polarization.The separation of the illumination spots of the two beams and theirorientation on the sample can be influenced directly by the adaptivemirrors 15 and 17. The beam paths are automatically corrected, i.e.,made to coincide on the detector, in the detection beam path whenpassing the adaptive mirrors 15 and 17. When using a polarizer with anaxis inclined by 45° to the polarization direction of the two beams infront of the detector, there is interference of the two beams on theconverters 27.1 and 27.2 which can be detected confocally ornonconfocally.

Fluorescence Microscopy

In this type of operation, the illumination light excites fluorescencein the sample which is used to generate images.

With respect to fluorescence excitation, particularly in biologicalsamples, the polarization of the light is of secondary importance.Typically, the sample can be excited with linearly polarized light whichcomes, for example, from the adaptive mirror 15. The fluorescenceradiation is generally unpolarized and is divided at the beam splitter19.

The same procedure as that already described with reference toreflection microscopy can be used to obtain the gradients of theradiation intensity and accordingly the direction of the optimization ofthe mirror geometry with one measurement. The correction is carried outonly on the detection side with different mirror settings, which isrequired for gradient determination because the excitation light passesover an adaptive element.

The image generation is carried out without losses with an identicaloptimized position of the mirrors 15 and 17; the signals of theconverters 27.1 and 27.2 are added.

The following procedure can be implemented in order to carry outdifferent corrections in the illumination beam path and in the detectionbeam path. The sample is again excited by linearly polarized lightwhich, for example, comes from adaptive mirror 15. The signal receivedby the converter 27.2 can be used to optimize the mirror geometry of theadaptive mirror 17 independent from the reception signal of theconverter 27.1 for optimizing the mirror shape of the adaptive mirror15, wherein two measurements with a new mirror position are now requiredto determine a gradient.

The image generation is carried out with the mirror positions that havebeen optimized in this way, wherein the excitation light passes viamirror 15 and the detection light passes via mirror 17.

FIG. 5 shows an example for the correction with imaging of a biologicalsample at a depth of 500 μm with a water immersion objective with an NAof 1.2. The excitation wavelength is 488 nm, the fluorescence wavelengthis 550 nm. The deviation of the index of refraction of the sample fromthe index of refraction of the water immersion layer is 1.38−1.33=0.05and leads to a Strehl ratio of 0.11 without chromatic aberration of theoptical system. With chromatic aberration (primary longitudinalchromatic aberration of the focusing microscope objective) whichcorresponds to a change in focus of about 0.6 μm of the fluorescenceradiation compared to the excitation radiation, the Strehl ratiocontinues to fall to about 0.06.

Without chromatic aberrations, the optimal correction is achieved withinabout 15 iterations, taking into account the first and second orderspherical aberration. With chromatic aberration and optimal identicalcorrection in the excitation beam path and fluorescence beam path, theoptimized Strehl ratio is only 0.2. With an independent correction inthe excitation beam path and fluorescence beam path which can be carriedout with the present invention, a Strehl ratio of 0.99 can be achievedin spite of chromatic aberration.

Laser Microscopy with Nonlinear Excitation

With nonlinear excitation (multiphoton excited fluorescence, higherharmonics generation), a resolution equivalent to confocal detection isachieved without the use of a pinhole for detection. When a pinhole isused, the correction of image errors is required only in theillumination beam path.

In this case, the adaptive mirror 17 can be replaced by a nonadaptivemirror and a quarter-wave plate as was already mentioned with referenceto FIG. 2. The reception signal of the detector 38 is then used directlyto optimize the mirror geometry of the adaptive mirror 39 in the mannerdescribed above, wherein two measurements (mirror positions) are againrequired for determining a gradient.

When a pinhole is used to further increase resolution, the correction inthe detection beam path is important and can be achieved in case offluorescence, as was described above with respect to fluorescencemicroscopy and shown with reference to FIG. 1. In particular, anindependent correction in the illumination beam path and detection beampath is possible in this way. Due to the typically large differencebetween the wavelengths of excitation radiation and fluorescenceradiation, an independent correction of this kind is required to enablethe use of a pinhole.

An advantage of the iterative regulation of the adaptive optics which isdescribed herein consists in that the actual wavefront forming by theadaptive elements need not be known exactly, since signal optimizationis achieved iteratively by means of a variation process. Due to thenecessity of passing through different adaptive positions (iterations)per optimization, care must be taken that movements of the sample,bleaching or damage to the sample can not occur during optimization.Bleaching of or damage to the sample can be minimized by reducing theintensity of the illumination radiation because the detectionsensitivity used in the solution according to the invention for theadaptive correction is the same as that used as for obtaining images.

During the iterative process for the adaptive correction, the increasingimprovement in the reception signal allows a further reduction in theillumination intensity. A suitably selected initial position of theadaptive element makes it possible to reduce the quantity of iterationsduring the process and consequently to introduce less energy into thesample.

For the optimization itself, the above-mentioned processes play asubordinate role because a gradient is determined with respect to theactual state of the sample in each instance and is used to control theoptimization process.

Relative movements of the sample and optical system can also becorrected when a small but structured portion of the sample is scannedduring optimization. The image information can be used for correctingthe movement of the sample (e.g., through cross-correlation).

The movement correction is achieved by means of the adaptive mirrors bydefocusing by a suitable amount and/or by means of the scanning device 7or 34 of the microscope arrangement. The possibility of correcting fastmovements of the sample is another possible application of the adaptiveoptics for image acquisition in general. The electronic analysis ofsuccessive images (e.g., for purposes of improving contrast at highfrequencies) is another option for judging the correction process bymeans of the adaptive mirrors.

While the foregoing description and drawings represent the presentinvention, it will be obvious to those skilled in the art that variouschanges may be made therein without departing from the true spirit andscope of the present invention.

Reference Numbers  1 illumination source  2 illumination light  3 sample 4 scanning optics  5 tube lens  6 microscope objective  7 scanningdevice  8 beam splitter  9 detection beam path 10 detection device 11pinhole optics 12 pinhole 13 collimating optics 14 wavelength filter 15adaptive mirror 16 arm 17 adaptive mirror 18 arm 19 beam splitter 20quarter-wave plate 21 zoom optics 22 quarter-wave plate 23 zoom optics24 lens 25 pupil plane 26 beam splitter 27.1, 27.2 converters 28illumination source 29 illumination light 30 scanning optics 31 tubelens 32 microscope objective 33 sample 34 scanning device 35 beamsplitter 36 detection beam path 37 pinhole optics 38 detector 39adaptive mirror 40 nonadaptive mirror 41, 42 arm 43 beam splitter 44, 45quarter-wave plate 46 zoom optics 48 illumination source 49 illuminationlight 49.1 beam component 50 splitter surface 51 transmittive area 52reflective area 53 concave mirror 54 lens 55 scanning optics 56 tubelens 57 microscope objective 58 sample 59 scanning device 60 beamsplitter 61 detection beam path 61.1 beam component 62 splitter surface63 concave mirror 64, 65 detector 66 wavelength filter 67 relay optics

1. A laser scanning microscope, with an adaptive optical device in themicroscope beam path, comprising: two adaptive reflective elements, atleast one of which is constructed as an adaptive optical element, bothof which being oriented with their reflector surface vertical to theoptical axes of the microscope beam path, each adaptive reflectiveelement having a reflective surface with a geometry that is changed by acontrol; a polarizing beam splitter having a splitter layer which islocated in the vertex of two orthogonal arms of the microscope beam pathor two orthogonal portions of a folded microscope beam path, wherein afirst adaptive element is associated with one arm and the other adaptiveelement is associated with a second arm, and a quarter-wave plate beinglocated in each arm between the beam splitter and adaptive reflectiveelement; and a detection device to which the detection light is directedand which is linked to the adaptive elements by evaluating and adjustingdevices; and wherein the detection light coming from the sample isreflected by at least one of the adaptive elements.
 2. The laserscanning microscope according to claim 1; wherein, in one arm, the lightcoming from the illumination source, depending on its polarizationstate: passes through the splitter layer and a quarter-wave plate to afirst adaptive element, is then reflected back by the reflector surfaceof the adaptive element to the splitter layer and is deflected to themicroscope objective; and/or is deflected in the direction of a secondadaptive element and, after passing through a quarter-wave plate,strikes the second adaptive element, is reflected back by the latter tothe splitter layer and passes through the latter to the microscopeobjective; wherein, in the other arm, the detection light coming from asample through the microscope objective, depending on its polarizationstate: passes through the splitter layer and a quarter-wave plate to thesecond adaptive element, is then reflected back by the reflector surfaceof the latter to the splitter layer and is deflected to the detectiondevice; and/or is deflected by the splitter layer in the direction ofthe first adaptive element and, after passing through a quarter-waveplate, strikes the first adaptive element, is reflected back by thelatter to the splitter layer and passes through the latter to thedetection device; and wherein the detection device has a secondpolarizing beam splitter and two optoelectronic converters which arecoupled with the adaptive elements by an evaluating and adjustingdevice, wherein, depending on its polarization state, the detectionlight passes through the beam splitter to the one optoelectronicconverter or is deflected by the splitter layer to the otheroptoelectronic converter.
 3. The laser scanning microscope according toclaim 1; wherein pinhole optics for focusing the detection light on apinhole and a lens for collimating the detection light are providedbetween the beam splitter and the detection device.
 4. A laser scanningmicroscope, with an adaptive optical device in the microscope beam path,comprising: an adaptive reflective element and a nonadaptive reflectiveelement, both of which being oriented with their reflector surfacevertical to the optical axis of the microscope beam path, the adaptivereflective element having a reflective surface with a geometry that ischanged by a control; a polarizing beam splitter having a splitter layerlocated in the vertex of two orthogonal arms of the microscope beam pathor two orthogonal portions of a microscope beam path which is foldedmultiple times, wherein the adaptive element is associated with one armand the nonadaptive element is associated with a second arm, and aquarter-wave plate being located in each arm between the beam splitterand the respective reflective element; and a detection device to whichthe detection light is directed and which is linked to the adaptiveelement via an evaluating and adjusting device.
 5. The laser scanningmicroscope according to claim 4; wherein, in one arm the light comingfrom the illumination source, depending on its polarization state:passes through the splitter layer and a quarter-wave plate to theadaptive element, is reflected back to the splitter layer by thereflector surface of the adaptive element and is deflected by thesplitter layer toward the microscope objective; and/or is deflected bythe splitter layer in the direction of the reflective element, isreflected back to the splitter layer by the reflective element afterpassing through a quarter-wave plate, and passes through the splitterlayer to arrive at the microscope objective; wherein, in the other arm,the light coming from a sample through the microscope objective,depending on its polarization state: passes through the splitter layerand a quarter-wave plate to the reflective element, is reflected back bythe latter to the splitter layer and arrives at the detection device;and/or is deflected by the splitter layer in the direction of theadaptive element and, after passing through a quarter-wave plate, isreflected back by the adaptive element to the splitter layer and arrivesat the detection device.
 6. The laser scanning microscope according toclaim 4; wherein each adaptive element is positioned in a pupil plane ofthe microscope beam path, and optics are provided in the beam pathbetween the beam splitter and the microscope objective for imaging theplane of the adaptive elements in a pupil plane of the microscopearrangement.
 7. The laser scanning microscope according to claim 6;wherein the pupil plane is identical to the reflecting surface of thescanning device and the scanning optics, the microscope objective and atube lens are positioned relative to one another in such a way that adiffraction-limited spot is generated in the sample, which spot isguided over the sample in lateral direction due to the scanningmovement.
 8. The laser scanning microscope according to claim 4; whereinmirrors with mirror surfaces that are adjustable in segments, diaphragmmirrors or reflective spatial light modulators are provided as adaptiveelements.
 9. The laser scanning microscope according to claim 4; whereinzoom optics are associated with at least one of the adaptive elementsand serve to adapt the aperture of this adaptive element to the apertureof the microscope objective.
 10. A laser scanning microscope, with anadaptive optical device, comprising: an adaptive concave mirror which isarranged in a pupil plane of the illumination beam path, an optical beamsplitter being associated with the adaptive concave mirror in anintermediate image plane and having a transmittive area and a reflectivearea in a splitter surface, wherein the illumination light is directedinitially to the splitter surface and from the reflective area of thelatter to the concave mirror, is focused by the concave mirror onto thetransmittive area and passes through the latter to the sample, theadaptive concave mirror having a mirror surface with a geometry that ischanged by a control; and/or an adaptive concave mirror which isarranged in a pupil plane of the detection beam path, an optical beamsplitter being associated with the adaptive concave mirror in anintermediate image plane and having a transmittive area and a reflectivearea in a splitter surface, wherein the detection light is directedinitially to the splitter surface and from its reflective area to theconcave mirror, is focused by the concave mirror onto the transmittivearea and passes through the latter to a detection device, the adaptiveconcave mirror having a mirror surface with a geometry that is changedby a control; and evaluating and adjusting devices which are connectedto the adaptive element in the illumination beam path and/or to theadaptive element in the detection beam path, wherein the output signalsof the detection device are used for adaptive regulation of thewavefront of the illumination light or detection light.
 11. The laserscanning microscope according to claim 10; wherein the detection lightcoming from the sample is branched off from the microscope beam path bya beam splitter which is provided for this purpose in the microscopebeam path.
 12. The laser scanning microscope according to claim 11;wherein a detector is arranged downstream of the light coming from theillumination source and/or the detection light coming from the sample,and the beam component falling through the transmittive area impinges onsaid detector.
 13. The laser scanning microscope according to claim 11;wherein the adaptive element is coupled with a device for displacementin the direction of the optical axis, said device likewise beingconnected to the evaluating and adjusting devices, wherein the beamexpansion of the illumination light is influenced by the displacement ofthe adaptive concave mirror as well as by the change in the focal lengthof the concave mirror surface.
 14. The laser scanning microscopeaccording to claim 10; wherein a point-scanning device is provided forscanning the sample, and the transmittive areas of the splitter surfaceare formed as circular or elliptical openings.
 15. The laser scanningmicroscope according to claim 14; wherein the splitter surface isinclined by 45° relative to the incident radiation.
 16. A laser scanningmicroscope according to claim 10; wherein a line-scanning device isprovided for scanning the sample, and the transmittive areas of thesplitter surface are formed as slit-shaped openings.
 17. The laserscanning microscope according to claim 10; wherein information about theintensity of the received light signal is available at the outputs ofthe detection devices, said information is compared in the evaluatingunit to information stored therein, an actuating signal is generatedfrom the determined difference of the information about the intensity ofthe received light signal at the outputs of the detection devices andthe information stored in the evaluating unit, and said actuating signalis used to reposition the reflector surfaces of the adaptive elements.18. The laser scanning microscope according to claim 17; wherein theinformation stored in the evaluating device is obtained fromsimultaneous or previous detection with a defined other position of theadaptive elements.